Embryonic stem cells (ESCs) derived from the inner cell mass of blastocyst stage embryos are a powerful in vitro model for cellular differentiation and a potential source of cells for regenerative therapies. A better understanding of the factors controlling differentiation is necessary to robustly direct ESCs to produce mature cell types for therapeutic purposes. In addition, perturbations in differentiation in vivo lead to defects in early embryos and failed pregnancies. We are focusing on elucidating the components and wiring of the ESC gene regulatory network, in order to better control ESC differentiation and gain a more complete understanding of early development. Although three classes of regulatory factors comprise the ESC GRN?transcription factors, epigenetic regulators, and RNAs?the functions of only the first two classes are understood to a degree. We recently uncovered a key role for a structural feature of the ESC epigenome, RNA/DNA hybrids (RDHs), in cell fate. We found that RDHs are necessary to maintain the differentiation potential of ESCs?cells with reduced RDHs showed poor differentiation fidelity and a skewed differentiation profile. We recently found that RDHs play a key role in the ESC GRN, which likely accounts for these phenotypes. Depletion of RDHs leads to misregulation of thousands of genes. Interestingly, for a small fraction of these genes, we found that RDHs regulate the binding of two key epigenetic regulatory factors, PRC2 and Tip60-p400. However, most genes regulated by RDHs are neither direct nor indirect targets of these factors, raising the question of what other components of the GRN are modulated by RDHs. Here we propose to use epigenomic profiling and systems level approaches to comprehensively elucidate the roles of RDHs in the GRN. In addition, we will utilize a novel method for identification of new RDH-binding factors. Finally, we will use single cell profiling techniques to elucidate how RDHs regulate cell fate on a cell-by-cell basis. These studies will provide multiple new insights into how RNAs function within the ESC GRN. In addition, these studies will enhance our understanding of how cells acquire specific fates during ESC differentiation.